Oligospermic infertility associated with an androgen receptor mutation that disrupts interdomain and coactivator (TIF2) interactions
Farid J. Ghadessy, Joyce Lim, Abdullah A.R. Abdullah, Valerie Panet-Raymond, Chee Keong Choo, Rose Lumbroso, Thein G. Tut, Bruce Gottlieb, Leonard Pinsky, Mark A. Trifiro, Eu Leong Yong
Farid J. Ghadessy, Joyce Lim, Abdullah A.R. Abdullah, Valerie Panet-Raymond, Chee Keong Choo, Rose Lumbroso, Thein G. Tut, Bruce Gottlieb, Leonard Pinsky, Mark A. Trifiro, Eu Leong Yong
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Article

Oligospermic infertility associated with an androgen receptor mutation that disrupts interdomain and coactivator (TIF2) interactions

Abstract

Structural changes in the androgen receptor (AR) are one of the causes of defective spermatogenesis. We screened the AR gene of 173 infertile men with impaired spermatogenesis and identified 3 of them, unrelated, who each had a single adenine→guanine transition that changed codon 886 in exon 8 from methionine to valine. This mutation was significantly associated with the severely oligospermic phenotype and was not detected in 400 control AR alleles. Despite the location of this substitution in the ligand-binding domain (LBD) of the AR, neither the genital skin fibroblasts of the subjects nor transfected cell types expressing the mutant receptor had any androgen-binding abnormality. However, the mutant receptor had a consistently (approximately 50%) reduced capacity to transactivate each of 2 different androgen-inducible reporter genes in 3 different cell lines. Deficient transactivation correlated with reduced binding of mutant AR complexes to androgen response elements. Coexpression of AR domain fragments in mammalian and yeast two-hybrid studies suggests that the mutation disrupts interactions of the LBD with another LBD, with the NH2-terminal transactivation domain, and with the transcriptional intermediary factor TIF2. These data suggest that a functional element centered around M886 has a role, not for ligand binding, but for interdomain and coactivator interactions culminating in the formation of a normal transcription complex.

Authors

Farid J. Ghadessy, Joyce Lim, Abdullah A.R. Abdullah, Valerie Panet-Raymond, Chee Keong Choo, Rose Lumbroso, Thein G. Tut, Bruce Gottlieb, Leonard Pinsky, Mark A. Trifiro, Eu Leong Yong

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Figure 7

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(a) Effect of TIF2 on AR activity in HeLa cells. Mutant or WT AR plasmid...
(a) Effect of TIF2 on AR activity in HeLa cells. Mutant or WT AR plasmids were cotransfected with the indicated amounts of cDNA encoding full-length TIF2, with (+) or without (–) 0.01 nM MB (left). Cells were transfected with 50 ng TIF2 cDNA and exposed to increasing doses of MB (right). AR activity was measured with a multimeric AR reporter gene (ARE-TATA-Luc). (b) Interactions of TIF2 and ARLBD fragments in the mammalian two-hybrid assay. The fusion proteins VP16AD-TIF2 and GAL4DBD-ARLBD were coexpressed in HeLa cells, and protein-protein interactions were measured with (17m)5-E1bTATA-Luc reporter plasmid. Amino acid positions of TIF2 and ARLBD fragments are numbered. Vertical bars within the TIF2 fragment indicate nuclear receptor–interacting box motifs (LXXLL), and numbers indicate the first L of each consensus motif. In the left panel, cells were exposed to increasing doses of MB; in the right panel, cells were exposed to increasing doses of the VP16AD-TIF2 expression vector. Data are expressed as fold increase in luciferase activity with or without 1 nM MB. Bottom panels show an immunoblot of WT (lanes 1–3) and mutant (lanes 4–6) GAL4DBB-ARLBD fusion proteins (∼51 kDa) from representative cell lysates (10 μg protein per lane) and specific [3H]MB-binding activity (fmol/mg protein) of cells transfected with WT or mutant GAL4DBB-ARLBD vector. (c) Effect of TIF2 on TAD-LBD interactions in the mammalian two-hybrid assay. WT or mutant GAL4DBD-ARLBD fusion protein was coexpressed with VP16AD-ARTAD in HeLa cells, and TAD-LBD interactions were measured with a (17m)5-E1bTATA-Luc reporter plasmid (as in b). The effect of cotransfecting increasing doses of a fourth vector, pSG5-TIF2, encoding the full-length TIF2 protein, was measured as fold increase in luciferase activity with and without 0.1 nM MB. Data were the mean ± SE of at least 3 replicates.